Schistosoma mansoni is one of the most common etiological agents of human schistosomiasis and is estimated to infect more than 83 million humans in 54 countries. Praziquantel (PZQ) is the least expensive, easiest to use and most readily available of all current anti-schistosomal drugs. One problem associated with PZQ treatment is that it does not kill schistosomes for a period of 2-4 weeks after they infect the host. A second potential problem is the presence of drug resistance traits in natural populations of worms. As yet, neither the molecule to which PZQ binds nor its mechanism of action have been identified. Here, we propose to employ two complementary approaches to resolve these issues. We will identify the molecular target of PZQ using an engineered PZQ probe containing a diazirine group to covalently cross link the drug to its target and an alkyne group to which a reporter tag can be attached using click chemistry. Probe bound target will then be identified by chemiluminescent detection. Total cell protein as well as different cellular protein fractions will be one source of binding targets for this assay. In addition, we will specifically target the voltage gated Ca2+ channel Cav21 and 2 subunits as well as enolase and glyceraldehyde-3-phosphate dehydrogenase as potential PZQ binding proteins. We also propose to exploit the fact that different life cycle stages of S. mansoni have differing susceptibilities to PZQ by employing microarrays to compare the transcriptomes of PZQ sensitive miracidia, cercariae and mature schistosomes with those of mother sporocysts and juvenile schistosomes which are relatively insensitive. Previous experience suggests that this should provide a small pool of candidate target genes for further study and may also help identify members of the biochemical pathway driven by the PZQ target. These experiments will be performed in the presence and absence of PZQ. We will confirm the correct target or biochemical pathway has been identified by RNA inhibition of gene expression in mature schistosomes which should abolish the PZQ sensitive phenotype. Among the first potential pathway components to be targeted will be calmodulin, myosin light chain kinase and death associated protein kinase which may play a role in PZQ associated tegumental disruption. Small molecule libraries of PZQ analogs will be used to define the pharmacophore of the molecular target of PZQ. Finally, we will investigate the molecular basis of variable sensitivity to PZQ in Kenyan field isolates of S. mansoni. S. mansoni derived from natural infections and with varied sensitivities to PZQ will be maintained in mice. The transcriptomes of these populations will be compared using microarrays in an effort to determine if variable sensitivity is based on mutations to the PZQ target gene or differences in the expression of either (i) the target, (ii) a downstream component of the biochemical pathway driven by the PZQ target or (iii) a PZQ detoxification/clearance mechanism. More explicit knowledge of the binding target of PZQ and the mechanism of action of the drug will help us to devise improved assays for monitoring the emergence of resistance.